Global microRNA expression profiling: curcumin (diferuloylmethane) alters oxidative stress-responsive microRNAs in human ARPE-19 cells.

PURPOSE
In recent years, microRNAs (miRNAs) have been reported to play important roles in a broad range of biologic processes, including oxidative stress-mediated ocular diseases. In addition, the polyphenolic compound curcumin has been shown to possess anti-inflammatory, antioxidant, anticancer, antiproliferative, and proapoptotic activities. The aim of this study was to investigate the impact of curcumin on the expression profiles of miRNAs in ARPE-19 cells exposed to oxidative stress.


METHODS
MiRNA expression profiles were measured in ARPE-19 cells treated with 20 μΜ curcumin and 200 μΜ H₂O₂. PCR array analysis was performed using web-based software from SABiosciences. The cytotoxicity of ARPE-19 cells was determined with the CellTiter-Blue cell viability assay. The effects of curcumin on potential miRNA targets were analyzed with quantitative real-time PCR and western blotting.


RESULTS
Curcumin treatment alone for 6 h had no effect on ARPE-19 cell viability. Incubation with H₂O₂ (200 µM) alone for 18 h decreased cell viability by 12.5%. Curcumin alone downregulated 20 miRNAs and upregulated nine miRNAs compared with controls. H₂O₂ downregulated 18 miRNAs and upregulated 29 miRNAs. Furthermore, curcumin pretreatment in cells exposed to H₂O₂ significantly reduced the H₂O₂-induced expression of 17 miRNAs. As determined with quantitative real-time PCR and western blotting, curcumin increased the expression of antioxidant genes and reduced angiotensin II type 1 receptor, nuclear factor-kappa B, and vascular endothelial growth factor expression at the messenger RNA and protein levels.


CONCLUSIONS
The results demonstrated that curcumin alters the expression of H₂O₂-modulated miRNAs that are putative regulators of antioxidant defense and renin-angiotensin systems, which have been reported to be linked to ocular diseases.

intracellular antioxidant, glutathione, regulates antioxidant enzymes, and scavenges ROS [10,11]. However, the mechanisms underlying the antioxidant activity of curcumin have not been completely delineated. Curcumin has also been studied as a cancer chemopreventive agent in various cancers [12].
In recent years, miRNAs have received greater attention in cancer and other research fields. These small, non-coding RNAs bind to the 3′ untranslated region of target messenger RNA (mRNA) and negatively regulate the expression of genes involved in development, differentiation, proliferation, apoptosis, and other important cellular processes. MiRNAs regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA. Curcumin regulates the expression of genes involved in regulating cellular signaling pathways, including vascular endothelial growth factor (VEGF), nuclear factor-kappa B (NF-κB), protein kinase B, mitogen-activated protein kinase (MAPK), and other pathways [13], and these signaling pathways could be regulated by miRNAs. In this study, we evaluated the effects of curcumin on protecting RPE cells from H 2 O 2 -induced oxidative stress and identify a potential mechanism. The expression of miRNAs can be measured with northern blot, primer extension assay, RNase protection assay, and global profiling methods [14]. In our investigation, we used a PCR array to profile miRNA expression and to evaluate the effect of curcumin on oxidatively stressed ARPE-19 cells. We hypothesize that curcumin may play an important role in protecting RPE cells from oxidative stress by differentially modulating the expression of miRNAs that putatively regulate the expression of antioxidant, proangiogenic, proliferative, and proinflammatory genes. Our study for the first time reveals that the modulation of miRNA expression may be an important mechanism underlying the biologic effect of curcumin in human RPE, and this approach could be applied as a potential strategy for preventing and treating oxidative stress-mediated ocular diseases such as AMD and diabetic retinopathy (DR).

METHODS
Cell culture: ARPE-19 cells purchased from American Type Culture Collection (ATCC; Manassas, VA) were cultured at 37 °C in 5% (v/v) of CO 2 in Dulbecco's modified Eagle's medium and Ham's F12 medium (DMEM/F12) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ ml of penicillin, and 100 µg/ml of streptomycin (Invitrogen, Gibco, Carlsbad, CA). The media were changed every 2-3 days. ARPE-19 cells were seeded in 12-well plates at 1.5×10 5 cells/well, cultured for 48 h, and then treated with curcumin (Sigma-Aldrich, St. Louis, MO) and H 2 O 2 (Sigma-Aldrich) alone for 6 h and 18 h, respectively. The effect of curcumin on H 2 O 2 -induced oxidative stress was also assessed, in which ARPE-19 cells were treated with curcumin for 6 h before H 2 O 2 insult for 18 h and then harvested for miRNAenriched total RNA or protein extraction. Cells treated with dimethyl sulfoxide (DMSO) were maintained as controls.
Determination of cell viability: The CellTiter-Blue viability assay (Promega Corp, Madison, WI) was used as the index for cell survival, which measures the ability of living cells to reduce a redox dye (resazurin) into a fluorescent dye (resorufin). The assay was performed according to the manufacturer's protocol, in which 96-well plates were seeded at 1×10 4 cells/well and incubated for 6 h for cells to attach to the surface. The ARPE-19 cells were then exposed to varying concentrations (1-50 μM) of curcumin for 6 h. In addition, the cell viability during various durations of exposure of 20 μM curcumin was measured. Cells were washed with phosphate buffered saline (PBS; 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.8), 100 μl of DMEM-F12 without serum was added to each well, and then 20 μl CellTiter-Blue reagent was added. The plates were then incubated at 37 °C for 2 h. The absorbance was recorded at 590 nm in the Synergy 2 Multi-Mode Microplate Reader (Winooski, VT), with the CellTiter-Blue reagent without cells as the blank. The optic density (OD) of the experimental and control samples were subtracted from that of the blank. Cell viability (%) was calculated according to the following formula: Percentage cell viability=(OD of the experimental samples/OD of the control)×100.
RNA isolation, quantitative real-time polymerase chain reaction, and microRNA polymerase chain reaction arrays: MiRNA-enriched total RNA was extracted from cultured ARPE-19 cells using the QIAzol and miRNeasy kit following the manufacturer's protocol (Qiagen, Valencia, CA). The concentration of total RNA and the RNA quality (260/280 absorbance ratio) of the samples were measured using a SmartSpec 300 Spectrophotometer (Bio-Rad, Hercules, CA). The first strand kit (Qiagen, cat # 331,401) was used to perform cDNA analysis. For each reaction, 0.8 μg of total RNA, extracted from ARPE-19 cells treated with H 2 O 2 and with or without curcumin, was submitted to reverse transcription, following the manufacturer's instructions (Qiagen). The RNA sample with miRNA reverse transcription (RT) enzyme mix was incubated at 37 °C for 2 h, and then the samples were heated at 95 °C for 5 min to degrade RNA and inactivate the reverse transcriptase. To measure miRNAs, the cDNA was diluted tenfold by adding RNase-free H 2 O. The resulting diluted cDNA was added to the RT 2 Real-Time SYBR Green qPCR Master Mix (Qiagen), which contained real-time PCR buffer, a high-performance HotStart DNA Taq polymerase, nucleotides, and SYBR Green dye. The ROX and fluorescein reference dyes were also included in the PCR master mix to normalize variation from well to well.
For PCR array analysis, aliquots of the mixture were placed in each well of a 96-well RT 2 miRNA profiler miFinder PCR array plate (Qiagen, MAH-001A) that contained a panel of primer sets for a thoroughly researched set of 88 pathwayor disease-focused miRNAs, plus four small nuclear RNA housekeeping (SNORD 44, 47, 48, and U6) assays. The plate also contained duplicate reverse transcription controls that test the efficiency of the RT 2 miRNA first strand kit (Qiagen) reaction with a primer set detecting the template synthesized from the kit's built-in miRNA external RNA control and duplicate positive controls that tested the efficiency of the PCR reaction itself using a predispensed artificial DNA sequence and the primer set that detected it. The qRT-PCR analysis was performed in MyiQ Cycler (Bio-Rad Laboratories Inc.) with 25 μl total volume containing diluted cDNA (1 μl per well) and 2X SYBR Green PCR Master Mix. The amplification conditions were the following: 10 min at 95 °C, 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The relative amount of each miRNA in PCR array analysis was normalized to an average of four small nuclear housekeeping genes. Heatmap or cluster analysis was conducted on the expression profiles of all four groups using SABiosciences (Frederick, MD) software.
For mRNA analysis, the isolation of total RNA from ARPE-19 cells and cDNA synthesis were performed using the RNeasy kit and the QuantiTect reverse transcription kit, respectively, according to the manufacturer's protocol (Qiagen). The qRT-PCR analysis was performed with a 25 μl total volume containing cDNA (2 μl from each sample), 1X QantiFast SYBR Green PCR Master Mix (Qiagen), and 300 nM gene-specific primers ( Table 1). The amplification conditions for mRNA qRT-PCR were the following: 5 min at 95 °C, 40 cycles at 95 °C for 10 s, and 60 °C for 30 s. Each sample was assayed in duplicate, and the experimental data were normalized to the expression levels of the housekeeping gene Hprt. The absence of non-specific products was confirmed with the analysis of the melt curves and electrophoresis in 2% agarose gels.
The expression levels of mRNA and miRNAs were measured using the threshold cycle (C t ). The C t is the fractional cycle number at which the fluorescence of each sample passes the fixed threshold. Briefly, the average ΔC t of each group was calculated with the following formula: ΔC t =average mRNA/miRNA C t -average of the housekeeping genes C t . ΔΔC t was calculated with ΔΔC t =ΔC t of the experimental group -ΔC t of the control group. The fold relationships in miRNA or gene expression among the tested samples were calculated using 2 −ΔΔCt [15]. The efficiency of reverse transcription in the PCR array was calculated with ΔC t =average ΔC t RTC -ΔC t PPC . The ΔC t value of the RT control For data normalization, after the striping procedure, β-actin protein was detected on the same membrane. For β-actin and NF-κB detection, antimouse immunoglobulin G was used as a secondary antibody (sc-2005, Santa Cruz Biotechnology) at 1:5,000 dilution for 1 h at room temperature.
In addition to two major groups, PCR revealed a third group in which miRNAs were downregulated by H 2 O 2 and curcumin. Out of 81 miRNAs examined and followed by two criteria (statistical significance and 2-FC), the expression of three H 2 O 2 -downregulated miRNAs (let-7i, miR-106b, and miR-128) was significantly downregulated by curcumin pretreatment.
Effect of curcumin on gene expression: We evaluated the effect of curcumin on the expression of catalase, GPx-s, AT 1 R, NF-κB, and VEGF-A at the mRNA and protein levels. Exposure of ARPE-19 cells to various concentrations of curcumin Figure 3. Volcano plot of significance against the relative expression differences between the control and treated groups (A-C). Each dot represents one of the 81 microRNAs (miRNAs) that was filtered and had detectable expression in either treatment. The X-axis displays log2-transformed signal intensity differences between the control group and the experimental group; the Y axis is the log-odds calculated according to the moderated Student t statistic test for differential expression between the control group and the treated group. The horizontal dashed line and the vertical lines represent significance threshold log-odds=2 and twofold expression differences, respectively. All spots above the horizontal dashed line are miRNAs that were identified as showing significant differential expression between the two treatments. (1-20 μM) for 6 h resulted in a concentration-dependent increase in catalase and GPx-s expression at the mRNA and protein levels ( Figure 7). The increase in catalase and GPx-s expression at concentrations of 10 μM and above was significantly different from the vehicle (ethanol)-treated cells (control, p<0.001). Compared with the control, a sublethal concentration of H 2 O 2 (200 μM) significantly induced (p<0.001) the expression of AT 1 R, NF-κB, and VEGF-A at the mRNA and protein levels ( Figure 8). Curcumin (20 μM) significantly reduced the expression of NF-κB (p<0.05) and VEGF-A (p<0.001) at the mRNA and protein levels. In addition, curcumin pretreatment significantly (p<0.001) attenuated the H 2 O 2 -induced expression of AT 1 R, NF-κB, and VEGF-A at the mRNA and protein levels (Figure 8), indicating that the activation of AT 1 R, NF-κB, and VEGF-A is mediated by a prooxidant mechanism.

DISCUSSION
This study explored the potential modulation of miRNAs by curcumin in ARPE-19 cells using PCR array and identified several H 2 O 2 -modulated miRNAs whose expression was altered by curcumin. Dietary polyphenolic components such as curcumin have been implicated in many biologic pathways involved in development, differentiation, apoptosis, proliferation, and cellular stress signaling [9,18,19]. These processes have been reported to be regulated by miRNAs [20][21][22]. Bioinformatic analysis showed that a single miRNA is capable of modulating the expression of more than 100 mRNA targets and more than 50% of human protein coding genes could be regulated by miRNAs [23]. Therefore, to investigate the functional aspects of miRNAs, array-based miRNA surveys and other high-throughput approaches are becoming increasingly popular in biologic sciences. To date, 1,527 human mature miRNAs have been reported (miRBase 18). However, the exact number of ocular miRNAs expressed in the human retina or RPE is not yet known. For the first time, we report the effect of curcumin on the expression profiles of miRNAs in ARPE-19 cells, a cellular model for human retinal pigment epithelium.
Curcumin or H 2 O 2 treatment significantly affected the levels of many miRNAs in ARPE-19 cells. In general, more miRNAs were upregulated than downregulated in response to H 2 O 2 treatment, while curcumin treatment primarily downregulated expression. Of the miRNAs that were affected by both treatments, the direction (up-or downregulation) was opposite in all cases except one, miR-142-3p. In addition, curcumin counteracted the upregulation of miR expression by H 2 O 2 treatment. H 2 O 2 treatment significantly upregulated miR-30b and miR-30d, two members of the miR-30 family, which is      Following two criteria of statistical significance (p<0.05) and fold change (FC) (≥ or ≤2), the quantitative real-time polymerase chain reaction (qRT-PCR) array revealed five microRNAs (miRNAs) that were upregulated in cells treated with 20 μM curcumin for 6 h. The miRNAs have been reported to target angiotensin II type 1 receptor (AT1R), nuclear factor-kappa B (NF-κB), platelet-derived growth factor β (PDGFβ), and vascular endothelial growth factor (VEGF). Data represent mean±SEM from three separate samples. Data represent mean±SEM from three separate samples. *p<0.05 versus control, **p<0.001 versus control, #p<0.05 versus H2O2. consistent with our previous results [17], and all five members of the family were downregulated by the curcumin treatments. The mechanism of ROS-mediated gene regulation of miR-30b and miR-30d seems to be different from that of the other three members of the family. In silico analysis suggests that the expression of miR-30b and miR-30d, but not the other three members of the family, is regulated by the promoter of the zinc finger and AT hook domain-containing (ZFAT) gene. The epigenetic [2] and transcription factor-mediated regulation [3] of miRNA genes may underlie the molecular mechanism of ROS-mediated regulation of miRNAs in ARPE-19 cells.
Curcumin and other dietary components have been reported to alter the expression profiles of miRNAs in other tissues. In the human pancreatic cell line, curcumin has been shown to significantly up-and downregulate 11 and 18 miRNAs, respectively [5]. Consistent with those data, in our study, curcumin upregulated miR-103, miR-22, and miR-23b and downregulated miR-195, miR-15b, miR-196, and miR-92. In the human multidrug-resistant adenocarcinoma cell line A549/DDP, curcumin altered miRNA expression and significantly downregulated the expression of miR-186 [24], a negative regulator of the proapoptotic purinergic P2X7 receptor [25], which is also consistent with our result.
Curcumin was shown to significantly downregulate the H 2 O 2 -induced expression of miR-302 cluster in ARPE-19 cells. MiR-302 has been reported to inhibit several epigenetic regulators, including AOF1/2, methyl-CpG binding proteins 1 and 2, and DNA (cytosine-5-)-methyltransferase 1, that induce global DNA demethylation and subsequently activate transcription factors Oct4, Sox2, and Nanog [26]. The treatment of ARPE-19 cells with H 2 O 2 in our experiment induced miR-26b, miR-15b, and miR-9, and that induction was significantly suppressed by curcumin. The oxidant-induced expression of these three miRNAs showed consistency with the result shown in ARPE-19 cells treated with a retinoic acid derivative (4HPR), which induces ROS generation [27]. MiR-21 has been shown to protect cardiac myocytes against H 2 O 2 -induced injury via targeting the programmed cell death protein 4 and activator protein-1 pathway [28]. Our data also showed that miR-21 was sensitive to H 2 O 2 stimulation, and expression of miR-21 was significantly downregulated by curcumin pretreatment. The miR-17-92 cluster is expressed in human retinoblastoma, and upon deletion of Rb family members, miR-17-92 overexpression leads to explosive development of retinoblastoma [29]. In our investigation, miR-17 and miR-92 of the cluster were induced by H 2 O 2 -mediated stress, whereas curcumin treatments significantly downregulated the expression of the cluster.
The actions of curcumin and resveratrol, a structurallyrelated polyphenolic compound, are similar in some respects [30,31]. Curcumin and resveratrol target many of the same signaling molecules, including NF-κB, B-cell lymphoma 2, B-cell lymphoma-extra large, Bim, and survivin [32]. Curcumin and resveratrol have antioxidant effects that protect ARPE-19 cells from cytotoxicity [7,33,34]. Although the effects of resveratrol on miRNA expression in RPE cells have not been reported to our knowledge, resveratrol affects miRNA expression in several other cell types, and miRNA regulation is increasingly thought of as a means of delivering the beneficial effects of resveratrol [35] A major challenge for retinal miRNA studies is identifying relevant genes and their downstream targets that regulate angiogenesis and increased vascular permeability, the major factors for wet AMD and DR [36]. AMD and DR are the leading causes of blindness. Oxidative stress-mediated increases of VEGF, vascular endothelial growth factor receptor, Ang II, AT 1 R, NF-κB, and transforming growth factor beta promote angiogenesis and increased vascular permeability; these are the well-recognized regulatory factors for wet AMD and proliferative DR. Our qRT-PCR and immunoblotting data showed that the expression of AT 1 R, VEGF, and NF-κB was strongly upregulated with H 2 O 2 -mediated oxidative stress at the mRNA and protein levels. However, curcumin not only reversed the H 2 O 2 -mediated expression but also significantly decreased their expression compared with control. In our investigation, curcumin significantly induced the expression of five miRNAs (miR-146a, miR-150, miR-155, miR-20a, miR-22, and miR-126) that target downstream molecules such as VEGF, NF-κB, PDGFβ, and endothelin 1. The mechanism of action of curcumin on the modulation of miRNA expression is not well understood. The altered expression of miRNAs can occur via several molecular mechanisms such as transcriptional regulation, post-transcriptional processing, genomic abnormalities [37], and regulation by epigenetic factors [38]. MiR-146a was shown to be transactivated by NF-κB, but also to inhibit NF-κB activation, showing negative feedback regulation on NF-κB activation [39]. VEGF was reported to induce miR-20a and miR-155 in human umbilical vein endothelial cells [40]. However, miR-20a was also reported to target VEGF, indicating negative feedback regulation of miR-20a on VEGF [41]. MiR-150 and miR-155 were also reported to regulate PDGFβ [42] and AT 1 R [43], respectively. The miR-23-27-24 clusters enhance angiogenesis and choroidal neovascularization in mice by repressing sprouty2 and Sema6a proteins, which negatively regulate MAPK and VEGFR2 signaling in response to angiogenic factors [44]. In our analysis, curcumin downregulated two members of the clusters, miR-23b and miR-27b, which were upregulated by H 2 O 2 -mediated oxidative stress.
In summary, we evaluated the effect of curcumin on the expression levels of miRNA in ARPE-19 cells in the presence of an oxidative environment. For the first time, we have shown that this polyphenolic compound can alter the expression profiles of H 2 O 2 -modulated miRNAs in this human RPE culture system. Modulation of miRNA expression may be an important mechanism in the pathogenesis of AMD and DR, and curcumin may provide a therapeutic approach for preventing and treating these oxidative stress-mediated diseases.